Monoamine oxidase (MAO) inhibitors and uses thereof

ABSTRACT

The present invention provides a group of tobacco alkaloids, tobacco extract, Yerbamaté extract, and an extract of chewing gum and lozenges which are modulators of monoamine oxidase (MAO) activity (i.e., compounds and substances which inhibit MAO enzyme and prevent its biological activity). The MAO inhibitors of the present invention can cause an increase in the level of norepinephrine, dopamine, and serotonin in the brain and other tissues, and thus can cause a wide variety of pharmacological effects mediated by their effects on these compounds. The MAO inhibitors of the present invention are useful for a variety of therapeutic applications, such as the treatment of depression, disorders of attention and focus, mood and emotional disorders, Parkinson&#39;s disease, extrapyramidal disorders, hypertension, substance abuse, smoking substitution, anti-depression therapy, eating disorders, withdrawal syndromes, and the cessation of smoking.

This application is a division of application Ser. No. 10/042,164, filedJan. 11, 2002, now U.S. Pat. No. 6,569,470, which is a division of Ser.No. 09/325,852, filed Jun. 4, 1999, now U.S. Pat. No. 6,350,479, whichclaims priority under 35 U.S.C. § 119(e) to Provisional Application No.60/088,117, filed Jun. 5, 1998.

FIELD OF THE INVENTION

The present invention relates to the novel use of compounds andsubstances which are capable of modulating monoamine oxidase (MAO)activity by inhibiting the MAO enzyme. The present invention alsorelates to MAO inhibitors and their therapeutic use as a drug or dietarysupplement in the treatment of various conditions or disorders,including psychiatric and neurological illnesses. More particularly, thepresent invention relates to the therapeutic use of tobacco alkaloids,Yerbamaté (Ilex paraguariensis) extract, or tobacco extracts to inhibitMAO activity to provide a treatment for various disorders or conditions.

BACKGROUND OF THE INVENTION

By inhibiting MAO activity, MAO inhibitors can regulate the level ofmonoamines and their neurotransmitter release in different brain regionsand in the body (including dopamine, norepinephrine, and serotonin).Thus, MAO inhibitors can affect the modulation of neuroendocrinefunction, respiration, mood, motor control and function, focus andattention, concentration, memory and cognition, and the mechanisms ofsubstance abuse. Inhibitors of MAO have been demonstrated to haveeffects on attention, cognition, appetite, substance abuse, memory,cardiovascular function, extrapyramidal function, pain andgastrointestinal motility and function. The distribution of MAO in thebrain is widespread and includes the basal ganglia, cerebral cortex,limbic system, and mid and hind-brain nuclei. In the peripheral tissue,the distribution includes muscle, the gastrointestinal tract, thecardiovascular system, autonomic ganglia, the liver, and the endocrinicsystem. The present invention overcomes the problems and limitations ofthe prior art by providing methods and systems.

MAO inhibition by other inhibitors have been shown to increase monoaminecontent in the brain and body. Regulation of monoamine levels in thebody have been shown to be effective in numerous disease statesincluding depression, anxiety, stress disorders, diseases associatedwith memory function, neuroendocrine problems, cardiac dysfunction,gastrointestinal disturbances, eating disorders, hypertension,Parkinson's disease, memory disturbances, and withdrawal symptoms.

It has been suggested that cigarette smoke may have irreversibleinhibitory effect towards monoamine oxidase (MAO). A. A. Boulton, P. H.Yu and K. F. Tipton, “Biogenic Amine Adducts, Monoamine OxidaseInhibitors, and Smoking,” Lancet, 1(8577): 114-155 (Jan. 16, 1988),reported that the MAO-inhibiting properties of cigarette smoke may helpto explain the protective action of smoking against Parkinson's diseaseand also observed that patients with mental disorders who smoke heavilydo not experience unusual rates of smoking-induced disorders. It wassuggested that smoking, as an MAO inhibitor, may protect againstdopaminergic neurotoxicity that leads to Parkinson's disease and thatthe MAO-inhibiting properties of smoking may result in ananti-depressive effect in mental patients.

L. A. Carr and J. K. Basham, “Effects of Tobacco Smoke Constituents onMPTP Induced Toxicity and Monoamine Oxidase Activity in the MouseBrain,” Life Sciences, 48:1173-1177 (Jan. 16, 1991), found thatnicotine, 4-phenylpyridine and hydrazine prevented the decrease indopamine metabolite levels induced by1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice, but therewas no significant effect on dopamine levels. Because tobacco smokeparticulate matter caused a marked inhibition of MAO A and MAO Bactivity when added in vitro, it was suggested that one or moreunidentified substances in tobacco smoke are capable of inhibiting brainMAO and perhaps altering the formation of the active metabolite of MPTP.

J. S. Fowler, N. D. Volkow, G. J. Wang, N. Pappas, and J. Logan,“Inhibition of Monoamine Oxidase B in the Brain of Smokers,” Nature(Lond), 379(6567):733-736 (Feb. 22, 1996), found that the brains ofliving smokers showed a 40% decrease in the level of MAO B relative tonon-smokers or former smokers. MAO inhibition was also reported as beingassociated with decreased production of hydrogen peroxide.

It has also been suggested that nicotine may not be the only constituentof tobacco responsible for tobacco addiction. J. Stephenson, “CluesFound to Tobacco Addiction,” Journal of the American MedicalAssociation, 275(16): 1217-1218 (Apr. 24, 1996), discussing the work ofFowler, et al., pointed out that the brains of living smokers had lessMAO B compared with the brains of nonsmokers or former smokers. MAO B isan enzyme involved in the breakdown of dopamine, which is apleasure-enhancing neurotransmitter. The results suggested that theinhibition of MAO B in the brains of smokers may make nicotine moreaddictive by slowing down the breakdown of dopamine, thereby boostingits levels. The findings provided an explanation as to why cigarettesmokers were less susceptible to developing Parkinson's disease.Further, the findings suggested that MAO inhibitors could be used forsmoking cessation.

K. R. R. Krishnan, “Monoamine Oxidase Inhibitors,” The AmericanPsychiatric Press Textbook of Pharmacology, American Psychiatric Press,Inc., Washington, D.C. 1995, pp. 183-193, suggest various uses formonoamine oxidase inhibitors. The uses include atypical depression,major depression, dysthymia, melancholia, panic disorder, bulimia,atypical facial pain, anergic depression, treatment-resistantdepression, Parkinson's disease, obsessive-compulsive disorder,narcolepsy, headache, chronic pain syndrome, and generalized anxietydisorder.

D. Nutt and S. A. Montgomery, “Moclobemide in the Treatment of SocialPhobia,” Int. Clin. Psychopharmacol, 11 Suppl. 3: 77-82 (Jun. 11, 1996),reported that moclobemide, a reversible MAO inhibitor, may be effectivein the treatment of social phobia.

I. Berlin, et al., “A Reversible Monoamine Oxidase A Inhibitor(Moclobemide) Facilitates Smoking Cessation and Abstinence in Heavy,Dependent Smokers,” Clin. Pharmacol. Ther., 58(4): 444-452 (October1995), suggested that a reversible MAO A inhibitor can be used tofacilitate smoking cessation.

U.S. Pat. No. 3,870,794 discloses the administering of small quantitiesof nicotine and nicotine derivatives to mammals, including humans, toreduce anger and aggressiveness and to improve task performance.

U.S. Pat. No. 5,276,043 discloses the administering of an effectiveamount of certain anabasine compounds, certain unsaturated anabasinecompounds, or unsaturated nicotine compounds to treat neurodegenerativediseases.

U.S. Pat. No. 5,516,785 disclose a method of using anabasine, and DMABanabasine for stimulating brain cholinergic transmission and a methodfor making anabasine.

U.S. Pat. Nos. 5,594,011, 5,703,100, 5,705,512, and 5,723,477 disclosemodulators of acetylcholine receptors.

Known irreversible MAO inhibitors also inhibit MAO in the stomach andliver as well as the brain. As a result, their use has been limitedbecause hypertensive crisis may occur when certain types of food (forexample, fermented foods) are ingested, thereby creating an adversedrug-food interaction. Tyramine, which has a pressor action and which isnormally broken down by the MAO enzymes, can be present in certainfoods.

Thus, it would be desirable to provide MAO inhibitors which areeffective, but less potent (i.e., those which provide an asymptoticeffect on MAO inhibition) than known MAO inhibitors, for thetreatment-of various conditions and disorders. It would also bedesirable to provide MAO inhibitors which are easily synthesized andwhich could be provided to patients as an “over the counter” medicationor dietary supplement.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to the discovery that certain tobaccoalkaloids or extracts, a certain tea plant extract, and a certainextract of tobacco extract-containing chewing gum and lozenges provideMAO-inhibiting effects. The present invention also relates to the use ofthese compounds or substances in the treatment of certain conditions anddisorders in mammals, including humans.

The compounds and substances of the present invention are capable ofinhibiting MAO activity in mammalian brain and peripheral tissue. Thesecompounds and substances act by increasing the concentration ofmonoamine compounds (norepinephrine, dopamine, and serotonin) in thebody and brain.

The present invention provides a method of treating certain medical,psychiatric and/or neurological conditions or disorders. In a firstembodiment of the invention, the method comprises administering aMAO-inhibiting effective amount of anabasine, anatabine or nornicotineto a mammal, particularly a human, for the treatment of medical,psychiatric and/or neurological conditions and disorders such as, butnot limited to, Alzheimer's disease, Parkinson's disease, majordepression, minor depression, atypical depression, dysthymia, attentiondeficit disorder, hyperactivity, conduct disorder, narcolepsy, socialphobia, obsessive-compulsive disorder, atypical facial pain, eatingdisorders, drug withdrawal syndromes and drug dependence disorders,including dependence from alcohol, opioids, amphetamines, cocaine,tobacco, and cannabis (marijuana), melancholia, panic disorder, bulimia,anergic depression, treatment-resistant depression, headache, chronicpain syndrome, generalized anxiety disorder, and other conditions inwhich alteration of MAO activity could be of therapeutic value.

In a second embodiment of the invention, the method comprisesadministering a MAO-inhibiting effective amount of an extract ofYerbamaté (Ilex paraguariensis) tea plant to a mammal, particularly ahuman, for the treatment of medical, psychiatric and/or neurologicalconditions and disorders such as, but not limited to, Alzheimer'sdisease, Parkinson's disease, major depression, minor depression,atypical depression, dysthymia, attention deficit disorder,hyperactivity, conduct disorder, narcolepsy, social phobia,obsessive-compulsive disorder, atypical facial pain, eating disorders,drug withdrawal syndromes and drug dependence disorders, includingdependence from alcohol, opioids, amphetamines, cocaine, tobacco, andcannabis (marijuana), melancholia, panic disorder, bulimia, anergicdepression, treatment-resistant depression, headache, chronic painsyndrome, generalized anxiety disorder, and other conditions in whichalteration of MAO activity could be of therapeutic value.

In a third embodiment of the invention, the method comprisesadministering a MAO-inhibiting effective amount of a tobacco extract toa mammal, particularly a human, for the treatment of medical,psychiatric and/or neurological conditions and disorders such as, butnot limited to, Alzheimer's disease, Parkinson's disease, majordepression, minor depression, atypical depression, dysthymia, attentiondeficit disorder, hyperactivity, conduct disorder, narcolepsy, socialphobia, obsessive-compulsive disorder, atypical facial pain, eatingdisorders, drug withdrawal syndromes and drug dependence disorders,including dependence from alcohol, opioids, amphetamines, cocaine,tobacco, and cannabis (marijuana), melancholia, panic disorder, bulimia,anergic depression, treatment-resistant depression, headache, chronicpain syndrome, generalized anxiety disorder, and other conditions inwhich alteration of MAO activity could be of therapeutic value.

In a fourth embodiment of the invention, the method comprisesadministering a MAO-inhibiting effective amount of an extract of gum andlozenges formulated with tobacco extract to a mammal, particularly ahuman, for the treatment of medical, psychiatric and/or neurologicalconditions and disorders such as, but not limited to, Alzheimer'sdisease, Parkinson's disease, major depression, minor depression,atypical depression, dysthymia, attention deficit disorder,hyperactivity, conduct disorder, narcolepsy, social phobia,obsessive-compulsive disorder, atypical facial pain, eating disorders,drug withdrawal syndromes and drug dependence disorders, includingdependence from alcohol, opioids, amphetamines, cocaine, tobacco, andcannabis (marijuana), melancholia, panic disorder, bulimia, anergicdepression, treatment-resistant depression, headache, chronic painsyndrome, generalized anxiety disorder, and other conditions in whichalteration of MAO activity could be of therapeutic value.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a plot of MAO inhibition versus time for anabasine.

FIG. 2 shows the inhibition of MAO A and MAO B for anabasine.

FIG. 3 shows a plot of MAO inhibition versus time for anatabine.

FIG. 4 shows the inhibition of MAO A and MAO B for anatabine.

FIG. 5 shows a plot of MAO inhibition versus time for nornicotine.

FIG. 6 shows the inhibition of MAO A and MAO B for nornicotine.

FIG. 7 shows a plot of MAO inhibition versus time for Yerbamaté.

FIG. 8 shows the inhibition of MAO A and MAO B for Yerbamaté.

FIG. 9 shows a plot of MAO inhibition versus time for tobacco extract.

FIG. 10 shows the inhibition of MAO A and MAO B for tobacco extract.

FIG. 11 shows a plot of MAO inhibition versus time for GUMSMOKE.

FIG. 12 shows a plot of MAO inhibition versus time for a lozengeextract.

DETAILED DESCRIPTION OF THE INVENTION

MAO is an important enzyme that plays a major role in the metabolictransformation of catecholamines and serotonin. Neurotransmitters fromthis group are metabolized by MAO, and thus their effect is decreased attheir receptor cites. MAO is important for the regulation of the levelsof dopamine, norepinephrine and serotonin.

Accordingly, inhibition of this major enzyme system will have majoreffects on the functions regulated by this compound.

In a first embodiment of the invention, the method comprisesadministering a MAO-inhibiting effective amount of anabasine, anatabineor nornicotine to a mammal, particularly a human, for the treatment ofmedical, psychiatric and/or neurological conditions and disorders suchas, but not limited to, Alzheimer's disease, Parkinson's disease, majordepression, minor depression, atypical depression, dysthymia, attentiondeficit disorder, hyperactivity, conduct disorder, narcolepsy, socialphobia, obsessive compulsive disorder, atypical facial pain, eatingdisorders, drug withdrawal syndromes and drug dependence disorders,including dependence from alcohol, opioids, amphetamines, cocaine,tobacco, and cannabis (marijuana), melancholia, panic disorder, bulimia,anergic depression, treatment-resistant depression, headache, chronicpain syndrome, generalized anxiety disorder, and other conditions inwhich alteration of MAO activity could be of therapeutic value.

Anabasine, anatabine and nornicotine are minor tobacco alkaloids. Thesecompounds are commercially available. However, they may be synthesizedaccording to known techniques or extracted directly from tobacco itself.

Preferably, anatabine is synthesized according to the method disclosedby N. M. Deo and P. A. Crooks, “Regioselective Alkylation ofN-(diphenylmethylidine)-3-(aminomethylpyridine: A Simple Route to MinorTobacco Alkaloids and Related Compounds,” 1137-1141 (11 Dec. 1995),which is incorporated herein by reference.

In addition, nornicotine is preferably synthesized according to themethod disclosed by S. Brandange and L. Lindblom, “N-Vinyl as N-HProtecting Group: A Convenient Synthesis of Myosmine,” Acta Chem.Scand., B30, No. 1, p. 93 (1976), which is also incorporated herein byreference.

In a second embodiment of the invention, the method comprisesadministering a MAO-inhibiting effective amount of an extract ofYerbamaté (Ilex paraguariensis) tea plant to a mammal, particularly ahuman, for the treatment of medical, psychiatric and/or neurologicalconditions and disorders such as, but not limited to, Alzheimer'sdisease, Parkinson's disease, major depression, minor depression,atypical depression, dysthymia, attention deficit disorder,hyperactivity, conduct disorder, narcolepsy, social phobia,obsessive-compulsive disorder, atypical facial pain, eating disorders,drug withdrawal syndromes and drug dependence disorders, includingdependence from alcohol, opioids, amphetamines, cocaine, tobacco, andcannabis (marijuana), melancholia, panic disorder, bulimia, anergicdepression, treatment-resistant depression, headache, chronic painsyndrome, generalized anxiety disorder, and other conditions in whichalteration of MAO activity could be of therapeutic value.

The Yerbamaté extract may be prepared by shredding the Yerbamatématerials, mixing the shredded materials with a water/ethanol (forexample, 1/1 by volume) solution in a mixture of about four leaves per10 ml of the water/ethanol mixture, extracting with continuous stirring,and then removing the solution from the Yerbamaté residue. The residuecan then be further extracted two more times with the same volume ofwater/ethanol mixture, and then the extracts may be combined andfiltered to remove the particulate Yerbamaté materials. The combinedextracts may then be subject to vacuum evaporation to yield theYerbamaté extract.

In a third embodiment of the invention, the method comprisesadministering a MAO-inhibiting effective amount of a tobacco extract toa mammal, particularly a human, for the treatment of medical,psychiatric and/or neurological conditions and disorders such as, butnot limited to, Alzheimer's disease, Parkinson's disease, majordepression, minor depression, atypical depression, dysthymia, attentiondeficit disorder, hyperactivity, conduct disorder, narcolepsy, socialphobia, obsessive-compulsive disorder, atypical facial pain, eatingdisorders, drug withdrawal syndromes and drug dependence disorders,including dependence from alcohol, opioids, amphetamines, cocaine,tobacco, and cannabis (marijuana), melancholia, panic disorder, bulimia,anergic depression, treatment-resistant depression, headache, chronicpain syndrome, generalized anxiety disorder, and other conditions inwhich alteration of MAO activity could be of therapeutic value.

The tobacco extract may be prepared by shredding tobacco leaves (forexample, processed tobacco obtained from STAR TOBACCO, INC.), mixing theshredded leaves with a water/ethanol (for example, 1/1 by volume)solution in a mixture of about four leaves per 10 ml of thewater/ethanol mixture, extracting with continuous stirring, and thenremoving the solution from the tobacco residue. The residue can then befurther extracted two more times with the same volume of water/ethanolmixture, and then the extracts may be combined and filtered to removethe particulate tobacco leaf material. The combined extracts may then besubject to vacuum evaporation to yield the tobacco extract.

In a fourth embodiment of the invention, the method comprisesadministering a MAO-inhibiting effective amount of an extract of chewinggum and lozenges formulated with tobacco extract to a mammal,particularly a human, for the treatment of medical, psychiatric and/orneurological conditions and disorders such as, but not limited to,Alzheimer's disease, Parkinson's disease, major depression, minordepression, atypical depression, dysthymia, attention deficit disorder,hyperactivity, conduct disorder, narcolepsy, social phobia,obsessive-compulsive disorder, atypical facial pain, eating disorders,drug withdrawal syndromes and drug dependence disorders, includingdependence from alcohol, opioids, amphetamines, cocaine, tobacco, andcannabis (marijuana), melancholia, panic disorder, bulimia, anergicdepression, treatment-resistant depression, headache, chronic painsyndrome, generalized anxiety disorder, and other conditions in whichalteration of MAO activity could be of therapeutic value.

The chewing gum and lozenges extract may be prepared by extracting fiveslices of GUMSMOKE chewing gum and NICOMINT lozenges (obtained from STARTOBACCO, INC.), which are formulated with tobacco extract, withdistilled water (50 ml) at room temperature for 12 hours, and thenremoving the undissolved gum substance by filtration.

The above compounds and substances were evaluated for their MAOinhibiting activity. Test results surprisingly showed that the compoundsand substances of the present invention all provided MAO inhibition. Itwas also discovered that the MAO inhibiting effects had a differentcharacter than for known MAO inhibitors in that they reached anasymptotic or ceiling effect, so that further increases in the dosebeyond maximal inhibition did not produce any further increase in theMAO inhibition. This asymptotic effect would provide many benefits. Forexample, the problems associated with previously known, irreversible MAOinhibitors, such as hypertensive effects, can be avoided. Furthermore,the inventive MAO inhibitors may be provided as an “over the counter”drug or dietary supplement in view of its safety and efficacy.

The MAO inhibitors of the present invention may be provided in formswell known to one skilled in the art. They may be formulated in apharmaceutically acceptable carrier, diluent or vehicle and administeredin effective amounts. They may be provided in the form of a capsule,pill, tablets, lozenge, gum, troches, suppositories, powder packets orthe like.

The determination of the effective amounts for a given treatment can beaccomplished by routine experimentation and is also well within theordinary skill in the art.

EXAMPLES

To determine the effectiveness of compounds and substances of thepresent invention, experiments were conducted as follows:

MAO Reaction:

The MAO activities of the compounds and substances were determined usingstandard reaction conditions as described in Halt, A., et al.,Analytical Biochemistry, 244:384-392 (1997).

Tissue Preparation:

Liver samples from cow or rat were obtained immediately after sacrifice.Liver was homogenized in a Polytron mechanical homogenizer in a ratio of1 gram of liver to 1 ml of potassium phosphate buffer (0.2 M at pH of7.6). Large membranes were removed by low speed centrifugation at 1000×gfor 15 minutes. The supernatant was removed from the pellet and usedimmediately for MAO activity assays or stored at 0 degrees Centigrade.Protein levels were determined in the liver homogenate by the Bradfordprotein reaction.

Reaction Conditions:

The standard reaction conditions were developed as a modification of thespectrophotometric assay using standard conditions (Halt, A., et al.,Analytical Biochemistry, 244:384-392 (1997)). Total MAO activity wasdetermined by incubating the liver preparations for 30 minutes at 37degrees Centigrade with a 1/1 dilution of a test fraction (compound orsubstance to be tested dissolved in distilled water) or controlcondition (water alone). This incubation allowed the test compound orsubstance to interact with the enzyme under physiological conditions.The final tissue concentration in the reaction mixture was 3.5 mg per100 ml.

Following the incubation with test compounds/substances or control, theMAO reactions were initiated and the reactions were incubated at 37degrees Centigrade. The reaction was initiated by mixing 150 μl ofpreincubated tissue with 150 μl of chromogenic solution (containing 10mM vanillic acid, 5 mM 4-amino antipyrene, 20 units/ml of peroxidase in0.2 M potassium phosphate buffer final concentration pH 7.6), 600 μl ofamine substrate (tyramine 500 micromolar), and 100 μl of distilled water(1 ml reaction volume). The standard reaction time was for 1 hour, butreaction times varied from 1 minute to 3 hours to evaluate the timecourse of the reaction in the presence or absence of test substance orcontrol. The reactions were terminated by the addition of 30 μl of astop solution of phenelzine (10 mM). The stopped reactions were storedon ice and placed at room temperature for reading in a spectrophotometerat a wavelength of 498 nm. The resulting values were analyzed todetermine the amount of reaction product produced by MAO activity. Thisassay was reliable and simple to perform. A standard curve usinghydrogen peroxide for enzyme activity was prepared for each experimentto determine the activity of the enzyme.

Selective assays of MAO A and MAO B isoforms were determined by usingselective inhibitors of each of these enzymes. During the preincubationof the enzyme with the test solutions, either pargyline or chlorgyline(final drug concentrations in the reaction mixture of 500 AM) was addedto the reaction mixture. This technique allowed for the assay of MAO Aor MAO B activity in the absence of the activity of the other isoform ofthe enzyme. All other reaction conditions were conducted as for totalMAO activity studies.

Each of the compounds and substances of the present invention wereevaluated by initially determining a concentration curve at a reactiontime of one hour. After determining the concentration curves of eachcompound or substance on MAO activity, a reaction time course in thepresence or absence of test compound or substance was determined andtime course curves were generated. Following these experiments, theeffect of each test compound or substance was evaluated on MAO A and MAOB activity by the same reaction studies as described above for the totalenzyme activity.

Example 1

Anabasine, in its purified form, was dissolved in distilled water in amaximal inhibition concentration of 0.2 mg/ml, and tested according tothe procedure described above. At maximal or saturating inhibitionconcentrations, anabasine was effective at inhibiting MAO activity byapproximately 10-13%, and was effective at inhibiting the enzyme at alltime points in the reaction.

FIG. 1 presents the means (plus or minus the standard errors of themeans) for the percent inhibition of MAO activity produced by saturatingconcentrations of anabasine over 60 minutes of MAO activity measured asdescribed above. Each data point represented the mean of 5determinations. All the data points shown in FIG. 1 were statistically,significantly different from the sham control at each time point tested(student t test, p<0.01), and were representative of multipleexperiments.

Since anabasine was an inhibitor of MAO activity, further studies wereconducted to evaluate if this agent was inhibiting MAO A or B activityusing the methods described above. Anabasine was found to inhibit bothMAO A and MAO B activity as shown in FIG. 2. FIG. 2 presents the means(plus or minus the standard errors of the means) for 5 determinationsfor the percent inhibition of MAO A and MAO B activity. The effects ofanabasine on both forms of MAO activity were statistically,significantly different from control enzyme conditions (student t test,p<0.05). The results demonstrate that anabasine inhibits both MAO A andB forms of the enzyme.

Example 2

Anatabine in its purified form, was dissolved in distilled water in amaximal inhibition concentration of 0.1 mg/ml, and tested according tothe procedure described above. At maximal or saturating inhibitionconcentrations, anatabine was effective at inhibiting MAO activity byapproximately 60%. This result shows that anatabine may be much safer asa medication than standard MAO enzyme inhibitors. Anatabine waseffective at inhibiting the enzyme at all time points in the reaction,and was equally effective in inhibiting both MAO A and MAO B activities.

FIG. 3 presents the means (plus or minus the standard errors of themeans) for the percent inhibition of MAO activity produced by saturatingconcentrations of anatabine over 60 minutes of MAO activity measured asdescribed above. Each data point represented the mean of 6determinations. Anatabine was an effective MAO inhibitor at maximalconcentrations, inhibiting the enzyme by approximately 60%, as discussedabove. All the data points shown in FIG. 3 were statistically,significantly different from the sham control at each time point tested(student t test, p<0.005) and were representative of multipleexperiments.

Since anatabine was an inhibitor of MAO activity, further studies wereconducted to evaluate if this agent was inhibiting MAO A or B activityusing the methods described above. Anatabine was found to inhibit bothMAO A and MAO B activity as shown in FIG. 4. FIG. 4 presents the means(plus or minus the standard errors of the means) for 6 determinationsfor the percent inhibition of MAO A and MAO B activity. The effects ofanatabine on both forms of MAO activity were statistically,significantly different from control enzyme conditions (student t test,p<0.01). The results demonstrate that anatabine inhibits both MAO A and-B forms of the enzyme.

Example 3

Nornicotine in its purified form, was dissolved in distilled water in amaximal inhibition concentration of 0.08 mg/ml, and tested according tothe procedure described above. At maximal or saturating inhibitionconcentrations, nornicotine was effective at inhibiting MAO activity byapproximately 80 to 95%, and was effective at inhibiting the enzyme atall time points in the reaction. Nornicotine was also equally effectivein inhibiting both MAO A and MAO B activities.

FIG. 5 presents the means (plus or minus the standard errors of themeans) for the percent inhibition of MAO activity produced by saturatingconcentrations of nornicotine over 60 minutes of MAO activity measuredas described above. Each data point represented the mean of 6determinations. Nornicotine was an effective MAO inhibitor at maximalconcentrations, inhibiting the enzyme by approximately 80-95%, asdiscussed above. All the data points shown in FIG. 5 were statistically,significantly different from the sham control at each time point tested(student t test, p<0.01) and were representative of multipleexperiments.

Since nornicotine was an inhibitor of MAO activity, further studies wereconducted to evaluate if this agent was inhibiting MAO A or B activityusing the methods described above. Nornicotine was found to inhibit bothMAO A and MAO B activity as shown in FIG. 6. FIG. 6 presents the means(plus or minus the standard errors of the means) for 6 determinationsfor the percent inhibition of MAO A and MAO B activity.

The effects of nornicotine on both forms of MAO activity werestatistically, significantly different from control enzyme conditions(student t test, p<0.01). The results demonstrate that nornicotineinhibits both MAO A and B forms of the enzyme.

Example 4

The Yerbamaté extract was prepared as follows: Yerbamaté materials(obtained from STAR TOBACCO, INC.) were shredded and mixed with awater/ethanol (1/1 by volume) solution in a mixture of about four leavesper 10 ml of the water/ethanol mixture; the materials were thenextracted overnight with continuous stirring; the solution was thenremoved from the Yerbamaté residue and stored; the residue was thenfurther extracted overnight two more times with the same volume ofwater/ethanol mixture, and the three extracts were combined and filteredto remove the particulate Yerbamaté material; and the combined extractswere subjected to removal of the water/ethanol by vacuum evaporation.The resultant extract was then weighed and solubilized in distilledwater.

When tested, Yerbamaté extract was effective in inhibiting MAO activity.The maximal inhibition concentration was 10 mg/ml. At maximal orsaturating inhibition concentrations, the Yerbamaté extract inhibitedMAO activity by approximately 40 to 50%. The results suggest thatYerbamaté may be much safer as a medication than standard MAO enzymeinhibitors. The extract was effective in inhibiting MAO at all timepoints in the reaction, and was equally effective in inhibiting both MAOA and MAO B activities.

FIG. 7 presents the means (plus or minus the standard errors of themeans) for the percent inhibition of MAO activity produced by saturatingconcentrations of Yerbamaté over 60 minutes of MAO activity measured asdescribed above. Each data point represented the mean of 5determinations. Yerbamaté was an effective MAO inhibitor at maximalconcentrations, inhibiting the enzyme by approximately 40-50%, asdiscussed above. All the data points shown in FIG. 7 were statistically,significantly different from the sham control at each time point tested(student t test, p<0.005) and were representative of multipleexperiments.

Since Yerbamaté was an inhibitor of MAO activity, further studies wereconducted to evaluate if this agent was inhibiting MAO A or B activityusing the methods described above: Yerbamaté was found to inhibit bothMAO A and MAO B activity as shown in FIG. 8. FIG. 8 presents the means(plus or minus the standard errors of the means) for 5 determinationsfor the percent inhibition of MAO A and MAO B activity.

The effects of Yerbamaté on both forms of MAO activity werestatistically, significantly different from control enzyme conditions(student t test, p<0.01). The results demonstrate that Yerbamatéinhibits both MAO A and B forms of the enzyme.

Example 5

The tobacco extract was prepared in the same manner as in Example 4,except that processed tobacco leaves (obtained from STAR TOBACCO, INC.)were substituted for the Yerbamaté materials.

When tested, the tobacco extract was effective in inhibiting MAOactivity. At maximal or saturating inhibition concentrations, thetobacco extract was able to inhibit MAO activity by approximately 60%.The results suggest that the extract may be much safer as a medicationthan standard MAO enzyme inhibitors. The tobacco extract was effectiveat inhibiting MAO at all time points in the reaction, and was equallyeffective in inhibiting both MAO A and MAO B activities.

FIG. 9 presents the means (plus or minus the standard errors of themeans) for the percent inhibition of MAO activity produced by saturatingconcentrations of tobacco extract over 60 minutes of MAO activitymeasured as described above. Each data point represented the mean of 8determinations. Tobacco extract was an effective MAO inhibitor atmaximal concentrations, inhibiting the enzyme by approximately 60%, asdescribed above. All the data points shown in FIG. 9 were statistically,significantly different from the sham control at each time point tested(student t test, p<0.001) and were representative of multipleexperiments.

Since tobacco extract was an inhibitor of MAO activity, further studieswere conducted to evaluate if this agent was inhibiting MAO A or Bactivity using the methods described above. Tobacco extract was found toinhibit both MAO A and MAO B activity as shown in FIG. 10. FIG. 10presents the means (plus or minus the standard errors of the means) for8 determinations for the percent inhibition of MAO A and MAO B activity.The effects of tobacco extract on both forms of MAO activity werestatistically, significantly different from control enzyme conditions(student t test, p<0.005). The results demonstrate that tobacco extractinhibits both MAO A and B forms of the enzyme.

Examples 6 and 7

The extract of GUMSMOKE chewing gum or lozenges was prepared as follows:five slices each of gum or lozenges, formulated with tobacco extract,were extracted with 50 ml of distilled water at room temperature for 12hours. The undissolved gum substance was removed by filtration. (Thelozenges dissolved completely.) Dilutions of these extracts wereprepared for evaluation.

The gum and lozenges extracts were effective in inhibiting MAO activity.At maximal or saturating concentrations, the extracts were able toinhibit MAO activity by approximately 50 to 60%.

FIG. 11 presents the means (plus or minus the standard errors of themeans) for the percent inhibition of MAO activity produced by saturatingconcentrations of an extract of GUMSMOKE chewing gum prepared asdescribed above over 60 minutes of MAO activity measured as describedabove. Each data point represented the mean of 4 determinations.GUMSMOKE extract was an effective MAO inhibitor at maximalconcentrations, inhibiting the enzyme by approximately 50-60%. All thedata points shown in FIG. 11 were statistically, significantly differentfrom the sham control at each time point tested (student t test, p<0.05)and were representative of multiple experiments.

FIG. 12 presents the means (plus or minus the standard errors of themeans) for the percent inhibition of MAO activity produced by saturatingconcentrations of an extract of the lozenge prepared as described aboveover 60 minutes of MAO activity measured as described above. Each datapoint represented the mean of 4 determinations.

The lozenge extract was an effective MAO inhibitor at maximalconcentrations, inhibiting the enzyme by approximately 50-60%. All thedata points shown in FIG. 12 were statistically, significantly differentfrom the sham control at each time point tested (student t test, p<0.05)and were representative of multiple experiments. Both MAO A and MAO Bwere also inhibited by these extracts.

1. A method of treating generalized anxiety disorder, the methodcomprising administering to a mammal in need thereof an effective amountof anatabine in a pharmaceutically acceptable carrier, diluent orvehicle.
 2. The method according to claim 1, wherein the mammal ishuman.
 3. The method according to claim 2, wherein the anatabine isadministered in an amount effective to inhibit monoamine oxidase (MAO)activity.
 4. The method according to claim 3, wherein the inhibition ofMAO activity is asymptotic.
 5. The method according to claim 2, whereinthe anatabine is administered in an amount effective to inhibitmonoamine oxidase A (MAO A) activity.